Many diseases and conditions can induce internal bleeding into body fluids or excretions, but such bleeding may not always be visually detectable or apparent to a patient or healthcare provider. For example, gastrointestinal tumors and parasite infections may lead to bleeding into feces; kidney and bladder tumors may lead to bleeding into urine; lung cancer may lead to bleeding into the plural cavity; thoracic wall cancer may lead to bleeding into the thoracic cavity; and a hemorrhage may cause bleeding into the brain. This visually non-detectable bleeding is referred to as occult blood. Occult bleeding can also refer to bleeding that is clinically evident but from an obscure source.
The ability to detect occult blood is very valuable because it can allow the early diagnosis of various diseases and conditions, including but not limited to gastrointestinal tumors, kidney tumors, bladder tumors, lung cancer, thoracic wall cancer, as well as parasite infestation. Importantly, early detection, diagnosis, and treatment for many of these disease and conditions can greatly increase a patient's chance of vurvival. Therefore, any improvement in the sensitivity or ease of use of a test that can detect occult blood in body fluids or excretions, as well as any reduction in costs associated with that assay, can potentially save lives.
Heme is a natural pigment which combines with the protein globin to form hemoglobin. Heme is an iron complex of a class of red pigments called porphyrins. Porphyrin exists in high amounts in erythrocytes (red blood cells), and itself has a very strong fluorescence when irraidated with the appropriate wavelength of light. The fluorescence of porphyrin, however, may be quenched when its rings structure is covalently bonded with ferric ion. An important feature of porphyrins is their ability to be metalated and demetalated. A number of metals can be inserted into the porphyrin cavity by using various metal salts, for example Fe, Zn, Cu, and Ni. Previous methods have used acids of various strengths to remove the metal (demetalation) from porphyrin, thereby causing it to fluoresce, for example in the HemoQuant® and Hemoccult® II tests.
The American Gastroenterological Association (AGA) proposes that occult bleeding is the initial presentation of a positive fecal occult blood test (FOBT) result and/or iron-deficiency anemia (IDA), when there is no evidence of visible blood loss to the patient or physician. Occult bleeding can result in chronic gastrointestinal (GI) blood loss, and is usually identified only by tests that detect fecal blood or, if bleeding is sufficient, when it manifests as iron deficiency. The AGA proposes that obscure bleeding is bleeding of unknown origin that persists or recurs (i.e., recurrent or persistent IDA, FOBT positivity, or visible bleeding) after a negative initial or primary endoscopy (colonoscopy and/or upper endoscopy) result. There are two clinical forms of obscure bleeding: 1) obscure-occult identified by recurrent IDA and/or recurrent positive FOBT, and 2) obscure-overt identified by recurrent passage of visible blood.
While the focus of fecal occult blood testing is the detection of colorectal cancer, there are many causes of occult GI bleeding. Any lesion can bleed into the GI tract leading to occult GI bleeding, including but not limited to epistaxis, bleeding gums, esophagitis, peptic ulcers, esophageal and gastric malignancies, hemobilia, angiodysplasia and other benign vascular malformations such as Osler-Weber-Rendu telangiectasias, benign colon polyps, inflammatory bowel disease, ischemic bowel disease, hemorrhoids, and anal fissures. Important lesions in the upper digestive tract may be detected during the evaluation of patients who test positively for occult blood or who have iron deficiency anemia, although there is no consensus on optimal strategies for evaluating the upper digestive tract during evaluation for occult bleeding.
A major problem with screening for colon cancer through the detection of occult blood is a high rate of false-positive results, leading to invasive and expensive additional testing for healthy individuals. Additionally, commercially available fecal occult blood tests have relatively low sensitivity and positive predictive value for occult blood detection and colon cancer screening. Despite the limitations of currently available fecal occult blood tests, annual screening is recommended by the United States Preventive Services Task Force (United States Preventive Services Task Force. Guide to Clinical Preventive Services, 2nd ed, Williams & Wilkins, Baltimore 1996), the World Health Organization (Winawer et al., Prevention of colorectal cancer: Guidelines based on new data. Bull World Health Organ 1995; 73:7), and the American Cancer Society (Byers et al., American Cancer Society guidelines for screening and surveillance for early detection of colorectal polyps and cancer. Update 1997. CA Cancer J Clin 1997; 47:154). Most screening programs are based on the detection of occult blood along with endoscopic or radiographic evaluation of the colon. This approach has been associated with up to a 33 percent reduction in mortality from colorectal cancer (Mandel et al., J Natl Cancer Inst 1999; 91:434).
Commercially available tests for occult blood include HemeSelect®, FECA®, Hemoccult® II, Hemoccult II Sense®, and HemoQuant®. The likelihood that these tests will detect gastrointestinal blood is affected by the anatomical level of bleeding, factors relating to the patient (such as stool transit time, stool mixing, and intraluminal hemoglobin degradation), and the intrinsic features of the bleeding of the GI tract lesion (e.g. irregular bleeding) (Ahlquist et al., Cancer 1989; 63:1826–30). HemeSelect® and FECA® are based on the immunologic recognition of intact human hemoglobin, and are relatively simple and inexpensive tests. While these tests appear to have greater specificity for bleeding sources in the colon, bleeding from upper GI sources may not be detected because as blood passes through the GI tract the hemoglobin may be sufficiently altered so that it is not recognized immunologically. Thus, while immunological tests have a theoretical advantage in terms of localizing bleeding to the lower GI tract, the use of the tests are limited by the inability to detect blood loss originating in the upper GI tract, loss of globin antigenicity at room temperature, and the requirement for laboratory processing.
Hemoccult® II and Hemoccult II Sensa® are guaiac-based fecal occult blood tests that make use of the pseudoperoxidase activity of hemoglobin, and have been widely used and extensively evaluated. Guaiac turns blue after oxidation by oxidants or peroxidases in the presence of an oxygen donor such as hydrogen peroxide. Since heme, either as intact hemoglobin or free heme, has pseudoperoxidase activity, it can be detected through the use of guaiac. Hemoccult® II is a widely used guaiac test for fecal occult blood, and Hemoccult II Sensa® is another guaiac test that is more sensitive to peroxidase-like materials (Allison et al., N Engl J Med 1996; 334:155–59). When either of these tests is used, patients are instructed to diet for at least two days and up to one week prior to the test. The diet typically involves no red meat or turnip/horseradish, no gastric irritant drugs, no aspirin or other nonsteroidal anti-inflammatory drugs, no vitamin C, and an increased intake of high fiber foods. Additionally, while fecal rehydration can markedly raise the sensitivity of these tests, it can also reduce specificity (Mandel et al., N Engl J Med 1993; 328:1365–71).
The likelihood that a guaiac-based test will be able to detect occult bleeding is generally proportional to the quantity of fecal heme, which in turn is related to the size and location of the bleeding lesion. Thus, these types of tests are generally best at detecting large, more distal lesions. But these guaiac-based tests are also inconsistent in their accuracy. For example, one study of the Hemoccult® II test found that fecal hemoglobin levels must exceed 10 mg per gram of stool (10 ml of daily blood loss) for the test to be positive 50 percent of the time, but stools containing hemoglobin levels of less than 1 mg per gram can result in a positive test (Stroehlein et al., Am J Dig Dis 1976; 21:841–44; Ahlquist, Approach to the patient with occult gastrointestinal bleeding. In: Yamada T, ed. Textbook of gastroenterology. 2nd ed. Vol. 1. Philadelphia: J. B. Lippincott, 1995: 699–717; incorporated herein by reference). Such data have raised questions about the accuracy of these types of tests for detecting colonic lesions (Lang and Ransohoff, JAMA 1994; 271:1011–13).
HemoQuant® is a fluorometric assay that measures heme and heme-derived porphyrin. In the upper gastrointestinal tract, hemoglobin is cleaved to form heme and globin. While some intraluminal heme (generally less than 15 percent) is reabsorbed in the small intestine, a portion of the remaining heme is converted to porphyrin and iron (“intestinal converted fraction” heme). This fraction cannot be detected by guaiac-based tests, but is detectable by HemoQuant®, which measures both heme and porphyrins and is therefore a highly accurate indicator of bleeding, regardless of whether the bleeding occurs in the upper, middle, or lower GI tract. Moreover, substances that may interfere with or cause false positive results for guaiac-based tests (e.g., vegetable peroxidases) do not affect the test. Another advantage of HemoQuant® is that, unlike the guaiac and immunologic tests, it can give quantitative assessments of blood loss. The main disadvantage of this test is that it is expensive and requires a more complicated laboratory technique, and therefore cannot be performed at the bedside or in the office.
Accordingly, the need exists for a simple test for occult blood that incorporates the advantages of the heme-porphyrin test and is free of the disadvantages of known guaiac-based and fluorescence-based tests. A simple and more economical test that would reduce the likelihood of false positive and negative readings would be a significant improvement in the art and could potentially save numerous lives.
Erythrocyte detection in tissues or blood vessels can also prove to be highly significant in the detection and treatment of cerebral vascular injuries.
When the main blood supply to a particular region of brain is abruptly stopped, the pathophysiological changes are different between the central and peripheral regions of the involved area. Acute neuronal death occurs within the first hour of initial loss of blood flow in the central region (ischemic core), whereas some of the neurons in peripheral regions (ischemic penumbra) undergo slow degeneration over a period of several hours-days (Hermann et al., Neuroscience 104, 947–955 (2001)). In contrast to the core, the penumbra undergoes dynamic changes throughout the ischemia and reperfusion process, and it may coalesce either with the ischemic core or the normal tissue depending on the reperfusion conditions. A significant therapeutic goal of clinical management in stroke patients is to salvage the viable tissue in this penumbra.
Local pre-ischemic circulatory conditions at the capillary level may not be fully restored for an extended period of time after blood flow is resumed in the major arteries, thereby rendering the penumbra volume vulnerable to a much longer period of partial ischemia. This condition has in the past been termed the “no-reflow” phenomenon (Ames et al., Am J Pathol. 52, 437–453 (1968)), and describes a microvasculature perfusion failure after cerebral ischemia and reperfusion.
Partial microcirculatory stasis after cerebral ischemia and reperfusion is a potential factor in delayed cell death in occluded blood vessels. Sometimes described as the “no-reflow” phenomenon, such partial microcirculatory stasis may contribute to the developing damage in ischemic penumbra region and lead to additional injury following reperfusion. Limitations in current detection techniques have left the extent and spatial distribution of the phenomenon undetermined, and have in fact raised questions as to its existence.
Accordingly, the need exists for a test that will establish the existence of the “no-reflow” phenomenon and provide a technique to establish the extent and spatial distribution of the phenomenon in connection with the diagnosis and treatment of subjects that are prone to, or have suffered from, cerebral vascular traume.